Journal of the American Association for Laboratory Animal Science Vol 59, No 3 Copyright 2020 May 2020 by the American Association for Laboratory Animal Science Pages 242–253 Review of Rodent Euthanasia Methods Nirah H Shomer,1,* Krystal H Allen-Worthington,2 Debra L Hickman,3 Mahesh Jonnalagadda,4 Joseph T Newsome,5 Andrea R Slate,6 Helen Valentine,7 Angelina M Williams,2 and Michele Wilkinson8 The optimal choice of euthanasia method for laboratory rodents depends on a number of factors, including the scientific goals of the study, the need to minimize animal pain and/or distress, applicable guidelines and laws, the training and profi- ciency of personnel, and the safety and emotional needs of the personnel performing the euthanasia. This manuscript aims to provide guidance to researchers so they may select the method of euthanasia that results in minimal experimental confounds, such as the creation of artifact and alteration of tissues and analytes. Specific situations addressed include euthanasia of large numbers of rodents and euthanasia of neonates. Recent literature supports the notion of significant strain-dependent differences in response to euthanasia methods such as CO2 inhalation. To assist researchers in selecting a strain-appropriate method of euthanasia, the authors present a summary of methodologies for assessing the effectiveness of euthanasia tech- niques, including elements and parameters for a scoring rubric to assess them. DOI: 10.30802/AALAS-JAALAS-19-000084 The goals of this review were to evaluate recent literature cohorts of rodents. This overview will discuss evaluation of related to rodent euthanasia and provide guidance and refer- the effectiveness of euthanasia techniques, including a scoring ences to help laboratory animal veterinarians make informed rubric to assess euthanasia techniques. decisions and recommendations related to humane and scientifi- cally appropriate techniques for rodent euthanasia. 63 Considerations for Choice of Euthanasia Method The AVMA Guidelines for the Euthanasia of Animals Compatibility with intended animal use. When euthanizing (AVMA Guidelines) is the primary reference in use at most US unwanted rodents (for example, retired breeders or pups of un- institutions. We used this document as a primary reference, wanted genotype) or study animals from whom terminal tissue and in addition, reviewed guidelines from other countries and collection is not needed, any of the approved methods in the regulatory bodies, and literature published since the last report. AVMA Guidelines may be suitable. The choice of method may Based on this new literature, veterinarians and institutions may be based on considerations such as minimizing emotional im- find it advisable to adopt methods that differ from the cur- pact on personnel, efficient workflow, occupational health and rent AVMA Guidelines. However, readers are reminded that safety considerations and logistics of convenience. However, if if an institution is required to comply with the Public Health terminal tissue collection is needed for diagnosis of clinical cases Service Policy, funded institutions must comply with the most or to collect data or tissues for a study, the veterinarian should recent published AVMA Guidelines. Protocol-specific, but not recommend a method that minimizes tissue artifact while program-wide, exemptions to this requirement are permissible, still providing a humane, painless death. Indeed, the AVMA but must be scientifically justified and approved by the Institu- Guidelines specify that “compatibility with intended animal tional Animal Care and Use Committee (IACUC). use and purpose and subsequent evaluation, examination, or Decisions on methods of euthanasia are complicated and use of tissue” should be a consideration in choosing a method. should be based on consultation with a laboratory animal vet- Tables 1 and 2 summarize literature citations comparing meth- erinarian. Veterinarians with advanced training or expertise in ods of euthanasia and their effects on a variety of measurements. Laboratory Animal Medicine (ACLAM or ECLAM or similar This is not an exhaustive list; many factors can affect the magnitude background and expertise) may be best suited to assist with and even directionality of differences between methods, including the choice and validation of an optimal technique. Recent lit- sex and age of the animals and handling techniques. The refer- 1,33,34,40,43,46,79 erature affirms that animals of different age, sex, ences in these tables provide a starting point for a more detailed disease state, and genetic background may respond differently literature search and for the design of pilot studies, as warranted. to euthanasia techniques. To ensure the method selected is The 2001 ANZCAART publication Euthanasia of Animals Used appropriate for the experimental animals and the aims of the for Scientific Purposes3 also contains an exhaustive review. The experimental protocol, a pilot study might be the best way to handling and restraint associated with euthanasia can also result ascertain the most appropriate euthanasia method for specific in sympathoadrenal activation, which can have profound ef- fects on certain analytes, tissue quality, and data reproducibility. Received: 11 Jun 2019. Revision requested: 05 Jul 2019. Accepted: 21 Oct 2019. Methods of euthanasia demonstrated in the literature to minimize 1Division of Comparative Medicine, Washington University in St Louis, St Louis, Mis- sympathoadrenal activation (for example, anesthesia) will not be souri; 2National Institutes of Health, NIMH, Bethesda, Maryland; 3Laboratory Animal Resource Center, Indiana University School of Medicine, Indianapolis, Indiana; 4Labora- effective if euthanasia is preceded by prolonged or rough handling tory Animal Medical Services, University of Cincinnati, Cincinnati Ohio; 5Division of or exposure of the animals to stressful stimuli. Laboratory Animal Resources and Department of Pathology, University of Pittsburgh, Effects on tissues. Euthanasia methods may affect tissues 6 Pittsburgh, Pennsylvania; Center for Comparative Medicine, Massachusetts General in a variety of ways. Physical methods such as decapitation Hospital, Boston, Massachusetts; 7Division of Animal Resources, University of Illinois, Urbana-Champaign, Urbana, Illinois; 8No affiliation and cervical dislocation offer the potential for a very quick *Corresponding author. Email: [email protected] death with no artifacts from chemical agents, but they also 242 Review of rodent euthanasia methods Table 1. Effects of euthanasia methods on cells and tissues (organized by system) System Method Compared with Affected parameters 49 Blood CD CD + SP, CO2, Isoflurane, or lymphocyte proliferation, cytotoxic lymphocyte halothane response, lymphocyte parameters, granulocyte, leukocyte, and platelet count Blood CD49,105 Antemortem/anesthetized murine bone marrow culture Brain CD58 CD + Isoflurane plasticity changes in brain slices. CD causes mechanical disruption/anatomic/morphologic changes 9,37 Brain Decapitation Decapitation+ CO2 GABA receptor function Cell viability and function CD49,105,113 Antemortem/anesthetized Normal lymphocyte proliferation Reproduction CD83 Isoflurane Isoflurane yielded fewer intact oocytes due to microhemorrhage that hindered collection 20 Reproduction CO2 Isoflurane Isoflurane reduced motile sperm counts in Sprague–Dawley rats, due to inhibition of vas deferens contractions and decrease in expelled sperm 44 Reproduction CO2 CD CO2 decreased fertilization rate of mouse oocytes Reproduction Decapitation92,97 *multiple – see references sperm motility, straight-line, average path, cur- vilinear velocities, linear index, and linearity 19 Pulmonary Argon CO2 No difference in pulmonary lesions. Note: Argon was aversive. 2,39,40,51,101 Pulmonary CO2 CO2 + O2 Lung: congestion, hemorrhage, emphysema, atelectasis; Cardiac muscle: variable degenera- tive changes (influenced by time of exposure to CO2 causing acidosis, hypoxia) Pulmonary Decapitation2,39,51,95 N/A Lung: emphysema, hemorrhage, blood in alveolar spaces Miscellaneous SP10,39,77,85 N/A Splenic enlargement due to smooth muscle re- laxation which lets spleen engorge with blood Miscellaneous SP1,33 Ethanol IP IP but not IV pentobarbital damages serosal cells; Pentobarbital and ethanol cause loss of tinctorial properties Metabolic Decapitation10 decapitation + anesthesia No significant differences in insulin and gluca- gon receptors from liver plasma membranes Heart muscle function CD85 SP Decreased coronary flow; decreased contractile function in isolated perfused heart preparations Heart muscle function Decapitation36 Antemortem/anesthesia rat heart mitochondrial function Smooth muscle function SP85 CD Decreased contractility in isolated muscle preps; Decreased GI smooth muscle contractility (PI or IV, not IP) Decreased spontaneous and drug induced vascular smooth muscle contractil- ity; IP only increased colonic contractility in response to acetylcholine Respiratory Decapitation39 Normal tissues marked focal accumulation of blood in bronchi- oles/alveolar spaces due to reflex inspiration Respiratory SP39 Normal tissues mild congestion of alveolar capillaries CD, Cervical Dislocation CO2, Carbon Dioxide Decapitation, Decapitation alone FBMI, Focused Microwave Beam Irradiation SP, Sodium Pentobarbital *, overdose with CO2, euthanasia solutions, enflurane, halothane, isoflurane, or sevoflurane, or decapitation after halothane, pentobarbital, or2 CO N/A, Not applicable cause tissue